Many of the functional proteins in living organisms are glycoproteins. It has been elucidated that the diversity of the sugar chains in glycoproteins play several important roles physiologically (Lain, R. A., Glycobiology, 4, 759-767, 1994).
In recent years, it has also become clear that the action of sugar chains can be divided into two categories. In the first case, sugar chains have a direct function as ligands for binding cells, or as receptors for bacteria and viruses, in the clearance of glycoproteins from the blood, lysosome targeting of lysosome enzymes and the targeting by glycoproteins toward specific tissues and organs. For example, the contribution of glycoprotein sugar chains in the infection of target cells by the AIDS virus (HIV) has been established (Rahebi, L. et al., Glycoconj. J., 12, 7-16, 1995). The surface of HIV is covered with envelope protein gp120. The binding of gp120 sugar chains to the CD4 of target cells is the beginning of infection by the HIV virus. In the second case, the sugar chain itself is not the functional molecule but indirectly contributes to the formation of the higher-order structure of proteins, solubility of proteins, protease resistance of proteins, inhibition of antigenicity, protein function modification, protein regeneration rate adjustment, and adjustment of the amount of proteins expressed in cell layers. For example, sugar chains are instrumental in the adjustment of the adhesion of nerve cell adhesion molecules which are distributed widely in the nervous system (Edelman, G. M., Ann. Rev. Biochem., 54, 135-169, 1985).
In eukaryotes, glycoprotein sugar chains are synthesized on lipids of the Endoplasmic reticulum as precursor sugar chains. The sugar chain portion is transferred to the protein, then some of the sugar residues on the protein are removed in the Endoplasmic reticulum, and then the glycoprotein is transported to Golgi bodies. In the Goldi bodies, after the excess sugar residues have been removed, further sugar residues (e.g. mannose) are added and the sugar chain is extended (Narimatsu, H., Microbiol. Immunol., 38, 489-504, 1994).
More specifically, for example, Glc3Man9GlcNAc2 on dolichol anchors is transferred to protein in the ER membrane (Moremen K. W., Trimble, R. B. and Herscovics A., Glycobiology 1994 April; 4(2):113-25, Glycosidases of the asparagine-linked oligosaccharide processing pathway: and Sturm, A. 1995 N-Glycosylation of plant proteins. In: New Comprehensive Biochemistry. Glycoproteins, Vol. 29a., Montreuil, J., Schachter, H. and Vliegenthart, J. F. G. (eds). Elsevier Science Publishers B.V., The Netherland, pp. 521-541). ER-glucosidase I and II removes three glucose units (Sturm, A. 1995, supra; and Kaushal G. P. and Elbein A. D., 1989, Glycoprotein processing enzymes in plants. In Methods Enzymology 179, Complex Carbohydrates Part F. Ginsburg V. (ed), Academic Press, Inc. NY, pp. 452-475). The resulting high mannose structure (Man9GlcNAc2) is trimmed by ER-mannosidase (Moremen K. W. et al, supra; and Kornfeld, R. and Kornfeld, S., Annu. Rev. Biochem. 54, 631-664, 1985; Assembly of asparagine-linked oligosaccharides). The number of mannose residues removed varies according to the differences in the accessibility to the processing enzymes. The isomers Man8-, Man7-, Man6- and Man5GlcNAc2 are produced during processing by ER-mannosidase and Mannosidase I (Kornfeld, R. and Kornfeld, S., supra). When four mannose residues are completely removed by Mannosidase I (Man I), the product is Man5GlcNAc2. N-acetylglucosaminyl transferase I (GlcNAc I) transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Man5GlcNAc2, resulting in GlcNAcMan5GlcNAc2 (Schachter, H., Narasimhan, S., Gleeson, P., and Vella, G., Glycosyltransferases involved in elongation of N-glycosidically linked oligosaccharides of the complex or N-acetylgalactosamine type. In: Methods Enzymol 98: Biomembranes Part L. Fleischer, S., and Fleischer, B. (ed), Academic Press, Inc. NY, pp. 98-134 pp. 98-134, 1983). Mannosidase II (Man II) removes two mannose residues from GlcNAcMan5GlcNAc2, yielding GlcNAcMan3GlcNAc2(Kaushal, G. P. and Elbein, A. D., supra; and Kornfeld, R. and Kornfeld, S., supra). The oligosaccharide GlcNAcMan4GlcNAc2 is used as a substrate of N-acetylglucosaminyl transferase II (GlcNAc II) (Moremen K. W. et al, supra; Kaushal, G. P. and Elbein, A. D., supra; and Kornfeld, R. and Kornfeld, S., supra). FIG. 19 summarizes the above described structures of N-linked glycans and enzymes involved in sugar chain modification pathway in the Endoplasmic reticulum and Goldi bodies. In FIG. 19, ⋄ denotes glucose, □ denotes GlcNAc, ◯ denotes mannose, ● denotes galactose, and ▪ denotes sialic acid, respectively.
The sugar addition in the Golgi bodies is called terminal sugar chain synthesis. The process differs widely among living organisms. The sugar chain synthesis depends on the type of eukaryote. The resulting sugar chain structure is species-specific, and reflects the evolution of sugar adding transferase and the Golgi bodies (Narimatsu, H., Cellular Biology, 15, 802-810, 1996).
Regarding aspargine-linked (N-linked) sugar chains; in animals, there are high mannose-type sugar chains, complex-type sugar chains and hybrid-type sugar chains. These structures are shown in FIG. 1. The complex-type sugar chains in plants have α1,3 fucose and β1,2 xylose which are sugar residues that are not found in animals (Johnson, K. D. and Chrispeels, M. J., Plant Physiol., 84, 1301-1308, 1897, Kimura, Y. et al., Biosci. Biotech. Biochem., 56, 215-222, 1992). In the case of N-linked sugar chains, sialic acid has been found in animal sugar chains but has not been found in plant sugar chains. Regarding galactose, which is generally found in animal sugar chains, although the presence thereof has been found in some plant sugar chains (Takahashi, N. and Hotta, T., Biochemistry, 25, 388-395, 1986), the examples thereof are few. The linkage-type thereof is β1,3 linkage (FEBS Lett 1997 Sep. 29, 415(2), 186-191, Identification of the human Lewis (a) carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium mytillus L.), Melo N S, Nimtz M, Contradt H S, Fevereiro P S, Costa J; Plant J. 1997 Dec. 12 (6), 1411-1417, N-glycans harboring the Lewis a epitope are expressed at the surface of plant cells. Fitchette-Laine A C, Gomord V, Cabanes M, Michalski J C, Saint Macary M, Foucher B, Cavelier B, Hawes C, Lerouge P, Faye L). This linkage is different from those found in animals.
Glycoproteins derived from humans include human erythropoietin (EPO). In order to produce glycoproteins with sugar chain structures similar to humans, these glycoproteins are produced in animal host cells. However, EPO produced in animal cells has a sugar chain structure that is different from the natural human sugar chain structure. As a result, in vivo activity of EPO is reduced (Takeuchi, M. et al., Proc. Natl. Acad. Sci. USA, 86, 7819-7822, 1989). The sugar chain structure in other proteins derived from humans, such as hormones and interferon, have also been analyzed and manufactured with the same glycosylation limitations.
The methods used to introduce exogenous genes to plants include the Agrobacterium method (Weising, K. et al., Annu. Rev. Genet., 22, 421, 1988), the electroporation method (Toriyama, K. et al., Bio/Technology, 6, 1072, 1988), and the gold particle method (Gasser, C. G. and Fraley, R. T., Science, 244, 1293, 1989). Albumin (Sijmons, P. C. et al., Bio/Technology, 8, 217, 1990), enkephalin (Vandekerckhove, J. et al., Bio/Technology, 7, 929, 1989), and monoclonal antibodies (Benvenulo, E. et al., Plant Mol. Biol., 17, 865, 1991 and Hiatt, A. et al., Nature, 342, 76, 1989) have been manufactured in plants. Hepatitis B virus surface antigens (HBsAg) (Mason, H. S. et al., Proc. Natl. Acad. Sci. USA., 89, 11745, 1992) and secretion-type IgA (Hiatt, A. and Ma, J. S. K., FEBS Lett., 307, 71, 1992) have also been manufactured in plant cells. However, when human-derived glycoproteins are expressed in plants, the sugar chains in the manufactured glycoproteins have different structures than the sugar chains in the glycoproteins produced in humans because the sugar adding mechanism in plants is different from the sugar adding mechanism in animals. As a result, glycoproteins do not have the original physiological activity and may be immunogenic in humans (Wilson, I. B. H. et al., Glycobiol., Vol. 8, No. 7, pp. 651-661, 1998).